Methods of Treatment of inflammation related conditions using pluripotent anti-inflammatory and metabolic Modulators

ABSTRACT

Methods of treating acute and chronic inflammatory conditions, tissue transplant rejection, and/or organ transplant rejection comprising administering to a subject in need thereof a therapeutically effective amount of a pluripotent anti-inflammatory and metabolic modulators optionally in combination with one or more secondary therapeutic agents and pharmaceutical compositions thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/408,459 filed on Oct. 14, 2016 and U.S. Provisional Application No.62/570,973 filed on Oct. 11, 2017.

BACKGROUND

Acute and chronic inflammation appears to underlie most, if not all, thechronic diseases of today, including cardiovascular disease, type 2diabetes, chronic kidney disease, Alzheimer's disease and cancer [1].However, classical anti-inflammatory drugs—including non-steroidalanti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs(SAIDs)—are not indicated as part of the regular treatment for thesediseases. Common treatments include anti-platelet agents, inhibitors ofangiotensin II, insulin sensitizers, HMG-CoA reductase inhibitors andbeta blockers. Moreover, classical NSAIDS and SAIDs did not show anybenefit, if not adverse effect, in the treatment of cardiovascular,metabolic, neurodegenerative, cancer and chronic kidney diseases [2].Thus, embodiments of the invention described herein encompassanti-inflammatory treatments of low grade chronic inflammation thatunderlie most of the chronic non-transmissible diseases of todays.

SUMMARY

One embodiment within the scope of the invention is a method of treatingacute and chronic inflammatory conditions comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof Formula I:

or a pharmaceutically acceptable salt thereof.

In another embodiment the invention is a method of treating inflammationrelated conditions comprising administering to a subject in need thereofa therapeutically effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt, and a secondary therapeuticagent.

One embodiment within the scope of the invention is a method of treatingtissue allograft rejection comprising administering to a subject in needthereof a therapeutically effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof.

In another embodiment within the scope of the invention is a method oftreating organ transplant rejection comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof Formula I:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the method of treating organ transplant rejectioncomprises the treatment of skin allograft rejection comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates adduct formation of SANA with β-mercaptoethanol(BME) and Gutathione (GSH).

FIG. 2 shows that the reaction between SANA and BME has a second orderrate constant.

FIG. 3 illustrates the effect of SANA on LPS-induced NF-κB/p65subcellular localization in THP-1 macrophages.

FIG. 4 shows the inhibition of NF-kB-dependent gene expression in humanmacrophages by SANA.

FIG. 5 shows that SANA is a more potent inhibitor of NF-kB dependentgene expression in these cells than salicylic acid.

FIGS. 6 and 7 show induction of phase two enzymes Nrf2/Keap 1-dependentgene expression by SANA but not by salicylic acid.

In FIGS. 8 and 9 show the inhibition of inflammasome in THP-1 cellsdifferentiated into macrophages (PMA 200 nM, 48 hs.) by SANA but not bysalicylic acid.

FIG. 10 shows the effect of SANA on AMPK phosphorylation in vivo.

FIG. 11 illustrates the pAMPK phosphorylation levels of SANA compared tosalicylic acid in mouse livers at dosage levels from about 100 mg/kg toabout 300 mg/kg.

In FIG. 12 illustrates the pAMPK phosphorylation levels of SANA comparedto salicylic acid in mouse livers at dosage levels from about 100 mg/kgto about 400 mg/kg.

FIG. 13 shows that SANA decreases LPS-induced Il-1b secretion into theperitoneum in vivo.

FIG. 14 shows that SANA reverses insulin resistance in HFD-induced obesemice.

FIG. 15 shows that SANA unexpectedly does not inhibit GAPDH activitywhile that is commonly observed with nitroalkenes.

FIG. 16 demonstrates that SANA treated skin allograft rejection betterthan the control group.

FIG. 17 illustrates that SANA treated skin allograft rejection betterthan salicylic acid.

DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth.

As used herein, the term “about” means plus or minus 5% of the numericalvalue of the number with which it is being used. Therefore, about 50%means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly to a subject, whereby the agentpositively impacts the target. “Administering” a composition may beaccomplished by, for example, injection, oral administration, topicaladministration, or by these methods in combination with other knowntechniques. Such combination techniques include heating, radiation,ultrasound and the use of delivery agents. When a compound is providedin combination with one or more other active agents (e.g. otheranti-atherosclerotic agents such as the class of statins),“administration” and its variants are each understood to includeconcurrent and sequential provision of the compound or salt and otheragents.

By “pharmaceutically acceptable” it is meant the carrier, diluent,adjuvant, or excipient must be compatible with the other ingredients ofthe formulation and not deleterious to the recipient thereof.

“Composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. Such term inrelation to “pharmaceutical composition” is intended to encompass aproduct comprising the active ingredient(s), and the inert ingredient(s)that make up the carrier, as well as any product which results, directlyor indirectly, from combination, complexation or aggregation of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, the pharmaceutical compositions ofthe present invention encompass any composition made by admixing acompound of the present invention and a pharmaceutically acceptablecarrier.

As used herein, the term “agent,” “active agent,” “therapeutic agent,”or “therapeutic” means a compound or composition utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. Furthermore, the term “agent,” “active agent,”“therapeutic agent,” or “therapeutic” encompasses a combination of oneor more of the compounds of the present invention.

A “therapeutically effective amount” or “effective amount” of acomposition is a predetermined amount calculated to achieve the desiredeffect, i.e., to inhibit, block, or reverse the activation, migration,proliferation, alteration of cellular function, and to preserve thenormal function of cells. The activity contemplated by the methodsdescribed herein includes both medical therapeutic and/or prophylactictreatment, as appropriate, and the compositions of the invention may beused to provide improvement in any of the conditions described. It isalso contemplated that the compositions described herein may beadministered to healthy subjects or individuals not exhibiting symptomsbut who may be at risk of developing a particular disorder. The specificdose of a compound administered according to this invention to obtaintherapeutic and/or prophylactic effects will, of course, be determinedby the particular circumstances surrounding the case, including, forexample, the compound administered, the route of administration, and thecondition being treated. However, it will be understood that the chosendosage ranges are not intended to limit the scope of the invention inany way. A therapeutically effective amount of compound of thisinvention is typically an amount such that when it is administered in aphysiologically tolerable excipient composition, it is sufficient toachieve an effective systemic concentration or local concentration inthe tissue.

The terms “treat,” “treated,” or “treating” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder, or disease, or to obtain beneficialor desired clinical results. For the purposes of this invention,beneficial or desired results include, but are not limited to,alleviation of symptoms; diminishment of the extent of the condition,disorder, or disease; stabilization (i.e., not worsening) of the stateof the condition, disorder, or disease; delay in onset or slowing of theprogression of the condition, disorder, or disease; amelioration of thecondition, disorder, or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder, or disease. Treatment includesprolonging survival as compared to expected survival if not receivingtreatment.

The term “subject,” as used herein, describes an organism, includingmammals, to which treatment with the compositions and compoundsaccording to the subject disclosure can be administered. Mammalianspecies that can benefit from the disclosed methods include, but are notlimited to, apes, chimpanzees, orangutans, humans, monkeys; and otheranimals such as dogs, cats, horses, cattle, pigs, sheep, goats,chickens, mice, rats, guinea pigs, and hamsters. Typically, the subjectis a human.

The term “tissue,” as used herein, describes an aggregate of cellstypically of a particular kind together with their intercellularsubstance that form one of the structural materials of a subject. Theterm “organ,” as used herein, describes a group of tissues that performa specific function. For example, skin is a type of organ embodiedherein.

Administration and Compositions

The compounds and pharmaceutically-acceptable salts thereof can beadministered by means that produces contact of the active agent with theagent's site of action. They can be administered by conventional meansavailable for use in conjunction with pharmaceuticals in a dosage rangeof 0.001 to 1000 mg/kg of mammal (e.g. human) body weight per day in asingle dose or in divided doses. One dosage range is 0.01 to 500 mg/kgbody weight per day orally in a single dose or in divided doses.Administration can be delivered as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, buttypically are administered with a pharmaceutically acceptable excipientselected on the basis of the chosen route of administration and standardpharmaceutical practice.

Compounds can be administered by one or more ways. For example, thefollowing routes may be utilized: oral, parenteral (includingsubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques), inhalation, buccal, sublingual, orrectal, in the form of a unit dosage of a pharmaceutical compositioncontaining an effective amount of the compound and optionally incombination with one or more pharmaceutically-acceptable excipients suchas stabilizers, anti-oxidants, lubricants, bulking agents, fillers,carriers, adjuvants, vehicles, diluents and other readily knownexcipients in standard pharmaceutical practice.

Liquid preparations suitable for oral administration (e.g. suspensions,syrups, elixirs and other similar liquids) can employ media such aswater, glycols, oils, alcohols, and the like. Solid preparationssuitable for oral administration (e.g. powders, pills, capsules andtablets) can employ solid excipients such as starches, sugars, kaolin,lubricants, binders, disintegrating agents, antioxidants and the like.

Parenteral compositions typically employ sterile water as a carrier andoptionally other ingredients, such as solubility aids. Injectablesolutions can be prepared, for example, using a carrier comprising asaline solution, a glucose solution or a solution containing a mixtureof saline and glucose. Further guidance for methods suitable for use inpreparing pharmaceutical compositions is provided in Remington: TheScience and Practice of Pharmacy, 21^(st) edition (Lippincott Williams &Wilkins, 2006).

Therapeutic compounds can be administered orally in a dosage range ofabout 0.001 to 1000 mg/kg of mammal (e.g. human) body weight per day ina single dose or in divided doses. One dosage range is about 0.01 to 500mg/kg body weight per day orally in a single dose or in divided doses.For oral administration, the compositions can be provided in the form oftablets or capsules containing about 1.0 to 500 mg of the activeingredient, particularly about 1, 5, 10, 15, 20, 25, 50, 75, 100, 150,200, 250, 300, 400, 500, and 750 mg of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Thespecific dose level and frequency of dosage for any particular patientmay be varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy. In view of the factors affecting the specific doselevel and frequency it is contemplated that the dose frequency can rangefrom multiple doses daily to monthly dosages. The preferred dosefrequency ranges from twice a day to every two weeks. A more preferreddose frequency ranges from twice a day to weekly. A most preferred dosefrequency ranges from twice a day to twice a week.

In the methods of various embodiments, pharmaceutical compositionsincluding the active agent can be administered to a subject in an“effective amount.” An effective amount may be any amount that providesa beneficial effect to the patient, and in particular embodiments, theeffective amount is an amount that may 1) prevent the subject fromexperiencing one or more adverse effects associated with a administeredagents, such as those used to diagnose, identify, and treat medicalconditions, 2) reduce side effects experienced by the subject as aresult of a medical therapy or reduce the side effects known to resultfrom such therapies, and/or 3) eliminate side effects resulting from amedical treatment experienced by the subject prior to administration ofthe active agent or eliminate the side effects known to result from suchtreatment. An effective amount may further be any amount that provides abeneficial effect to the patient, and in particular embodiments, theeffective amount is an amount that may 1) prevent or reduce rejection oftissue allografts and/or 2) prevent or reduce rejection of atransplanted organ.

Pharmaceutical formulations containing the compounds of the inventionand a suitable carrier can be in various forms including, but notlimited to, solids, solutions, powders, fluid emulsions, fluidsuspensions, semi-solids, and dry powders including an effective amountof an the active agent of the invention. It is also known in the artthat the active ingredients can be contained in such formulations withpharmaceutically acceptable diluents, fillers, disintegrants, binders,lubricants, surfactants, hydrophobic vehicles, water soluble vehicles,emulsifiers, buffers, humectants, moisturizers, solubilizers,antioxidants, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman& Gilman's, The Pharmaceutical Basis of Therapeutics, 6th Edition,MacMillan Publishing Co., New York (1980) both of which are herebyincorporated by reference in their entireties can be consulted.

Other embodiments of the invention include the active agent prepared asdescribed above which are formulated as a solid dosage form for oraladministration including capsules, tablets, pills, powders, andgranules. In such embodiments, the active compound may be admixed withone or more inert diluent such as sucrose, lactose, or starch. Suchdosage forms may also comprise, as in normal practice, additionalsubstances other than inert diluents, e.g., lubricating agents such asmagnesium stearate. In the case of capsules, tablets, and pills, thedosage forms may also comprise buffering agents and can additionally beprepared with enteric coatings.

In another exemplary embodiment, an oily preparation of an active agentprepared as described above may be lyophilized to form a solid that maybe mixed with one or more pharmaceutically acceptable excipient, carrieror diluent to form a tablet, and in yet another embodiment, the activeagent may be crystallized to from a solid which may be combined with apharmaceutically acceptable excipient, carrier or diluent to form atablet.

The means and methods for tableting are known in the art and one ofordinary skill in the art can refer to various references for guidance.For example, Pharmaceutical Manufacturing Handbook: Production andProcesses, Shayne Cox Gad, John Wiley & Sons, Inc., Hoboken, N.J.(2008), which is hereby incorporated by reference in its entirety can beconsulted.

Further embodiments which may be useful for oral administration of theactive agent include liquid dosage forms. In such embodiments, a liquiddosage may include a pharmaceutically acceptable emulsion, solution,suspension, syrup, and elixir containing inert diluents commonly used inthe art, such as water. Such compositions may also comprise adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable diluents include, but are notlimited to those described below:

Vegetable oil: As used herein, the term “vegetable oil” refers to acompound, or mixture of compounds, formed from ethoxylation of vegetableoil, wherein at least one chain of polyethylene glycol is covalentlybound to the vegetable oil. In some embodiments, the fatty acids mayhave between about twelve carbons to about eighteen carbons. In someembodiments, the amount of ethoxylation can vary from about 2 to about200, about 5 to 100, about 10 to about 80, about 20 to about 60, orabout 12 to about 18 of ethylene glycol repeat units. The vegetable oilmay be hydrogenated or unhydrogenated. Suitable vegetable oils include,but are not limited to castor oil, hydrogenated castor oil, sesame oil,corn oil, peanut oil, olive oil, sunflower oil, safflower oil, soybeanoil, benzyl benzoate, sesame oil, cottonseed oil, and palm oil. Othersuitable vegetable oils include commercially available synthetic oilssuch as, but not limited to, Miglyol™ 810 and 812 (available fromDynamit Nobel Chemicals, Sweden) Neobee™ M5 (available from DrewChemical Corp.), Alofine™ (available from Jarchem Industries), theLubritab™ series (available from JRS Pharma), the Sterotex™ (availablefrom Abitec Corp.), Softisan™ 154 (available from Sasol), Croduret™(available from Croda), Fancol™ (available from the Fanning Corp.),Cutina™ HR (available from Cognis), Simulsol™ (available from CJPetrow), EmCon™ CO (available from Amisol Co.), Lipvol™ CO, SES, andHS-K (available from Lipo), and Sterotex™ HM (available from AbitecCorp.). Other suitable vegetable oils, including sesame, castor, corn,and cottonseed oils, include those listed in R. C. Rowe and P. J.Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which isincorporated herein by reference in its entirety. Suitablepolyethoxylated vegetable oils, include but are not limited to,Cremaphor™ EL or RH series (available from BASF), Emulphor™ EL-719(available from Stepan products), and Emulphor™ EL-620P (available fromGAF).

Mineral oils: As used herein, the term “mineral oil” refers to bothunrefined and refined (light) mineral oil. Suitable mineral oilsinclude, but are not limited to, the Avatech™ grades (available fromAvatar Corp.), Drakeol™ grades (available from Penreco), Sirius™ grades(available from Shell), and the Citation™ grades (available from AvaterCorp.).

Castor oils: As used herein, the term “castor oil,” refers to a compoundformed from the ethoxylation of castor oil, wherein at least one chainof polyethylene glycol is covalently bound to the castor oil. The castoroil may be hydrogenated or unhydrogenated. Synonyms for polyethoxylatedcastor oil include, but are not limited to polyoxyl castor oil,hydrogenated polyoxyl castor oil, microgolglyceroli ricinoleas,macrogolglyceroli hydroxystearas, polyoxyl 35 castor oil, and polyoxyl40 hydrogenated castor oil. Suitable polyethoxylated castor oilsinclude, but are not limited to, the Nikkol™ HCO series (available fromNikko Chemicals Co. Ltd.), such as Nikkol HCO-30, HC-40, HC-50, andHC-60 (polyethylene glycol-30 hydrogenated castor oil, polyethyleneglycol-40 hydrogenated castor oil, polyethylene glycol-50 hydrogenatedcastor oil, and polyethylene glycol-60 hydrogenated castor oil,Emulphor™ EL-719 (castor oil 40 mole-ethoxylate, available from StepanProducts), the Cremophore™ series (available from BASF), which includesCremophore RH40, RH60, and EL35 (polyethylene glycol-40 hydrogenatedcastor oil, polyethylene glycol-60 hydrogenated castor oil, andpolyethylene glycol-35 hydrogenated castor oil, respectively), and theEmulgin® RO and HRE series (available from Cognis PharmaLine). Othersuitable polyoxyethylene castor oil derivatives include those listed inR. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients,(2006), 5th ed., which is incorporated herein by reference in itsentirety.

Sterol: As used herein, the term “sterol” refers to a compound, ormixture of compounds, derived from the ethoxylation of sterol molecule.Suitable polyethoyxlated sterols include, but are not limited to, PEG-24cholesterol ether, Solulan™ C-24 (available from Amerchol); PEG-30cholestanol, Nikkol™ DHC (available from Nikko); Phytosterol, GENEROL™series (available from Henkel); PEG-25 phyto sterol, Nikkol™ BPSH-25(available from Nikko); PEG-5 soya sterol, Nikkol™ BPS-5 (available fromNikko); PEG-10 soya sterol, Nikkol™ BPS-10 (available from Nikko);PEG-20 soya sterol, Nikkol™ BPS-20 (available from Nikko); and PEG-30soya sterol, Nikkol™ BPS-30 (available from Nikko).

Polyethylene glycol: As used herein, the term “polyethylene glycol” or“PEG” refers to a polymer containing ethylene glycol monomer units offormula —O—CH₂—CH₂—. Suitable polyethylene glycols may have a freehydroxyl group at each end of the polymer molecule, or may have one ormore hydroxyl groups etherified with a lower alkyl, e.g., a methylgroup. Also suitable are derivatives of polyethylene glycols havingesterifiable carboxy groups. Polyethylene glycols useful in the presentinvention can be polymers of any chain length or molecular weight, andcan include branching. In some embodiments, the average molecular weightof the polyethylene glycol is from about 200 to about 9000. In someembodiments, the average molecular weight of the polyethylene glycol isfrom about 200 to about 5000. In some embodiments, the average molecularweight of the polyethylene glycol is from about 200 to about 900. Insome embodiments, the average molecular weight of the polyethyleneglycol is about 400. Suitable polyethylene glycols include, but are notlimited to polyethylene glycol-200, polyethylene glycol-300,polyethylene glycol-400, polyethylene glycol-600, and polyethyleneglycol-900. The number following the dash in the name refers to theaverage molecular weight of the polymer. In some embodiments, thepolyethylene glycol is polyethylene glycol-400.

Suitable polyethylene glycols include, but are not limited to theCarbowax™ and Carbowax™ Sentry series (available from Dow), the Lipoxol™series (available from Brenntag), the Lutrol™ series (available fromBASF), and the Pluriol™ series (available from BASF).

Propylene glycol fatty acid ester: As used herein, the term “propyleneglycol fatty acid ester” refers to a monoether or diester, or mixturesthereof, formed between propylene glycol or polypropylene glycol and afatty acid. Fatty acids that are useful for deriving propylene glycolfatty alcohol ethers include, but are not limited to, those definedherein. In some embodiments, the monoester or diester is derived frompropylene glycol. In some embodiments, the monoester or diester hasabout 1 to about 200 oxypropylene units. In some embodiments, thepolypropylene glycol portion of the molecule has about 2 to about 100oxypropylene units. In some embodiments, the monoester or diester hasabout 4 to about 50 oxypropylene units. In some embodiments, themonoester or diester has about 4 to about 30 oxypropylene units.Suitable propylene glycol fatty acid esters include, but are not limitedto, propylene glycol laurates: Lauroglycol™ FCC and 90 (available fromGattefosse); propylene glycol caprylates: Capryol™ PGMC and 90(available from Gatefosse); and propylene glycol dicaprylocaprates:Labrafac™ PG (available from Gatefosse).

Stearoyl macrogol glyceride: Stearoyl macrogol glyceride refers to apolyglycolized glyceride synthesized predominately from stearic acid orfrom compounds derived predominately from stearic acid, although otherfatty acids or compounds derived from other fatty acids may be used inthe synthesis as well. Suitable stearoyl macrogol glycerides include,but are not limited to, Gelucire® 50/13 (available from Gattefosse).

In some embodiments, the diluent component comprises one or more ofmannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powderedcellulose, microcrystalline cellulose, carboxymethylcellulose,carboxyethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodiumstarch glycolate, pregelatinized starch, a calcium phosphate, a metalcarbonate, a metal oxide, or a metal aluminosilicate.

Exemplary excipients or carriers for use in solid and/or liquid dosageforms include, but are not limited to:

Sorbitol: Suitable sorbitols include, but are not limited to,PharmSorbidex E420 (available from Cargill), Liponic 70-NC and 76-NC(available from Lipo Chemical), Neosorb (available from Roquette),Partech SI (available from Merck), and Sorbogem (available from SPIPolyols).

Starch, sodium starch glycolate, and pregelatinized starch include, butare not limited to, those described in R. C. Rowe and P. J. Shesky,Handbook of Pharmaceutical Excipients, (2006), 5th ed., which isincorporated herein by reference in its entirety.

Disintegrant: The disintegrant may include one or more of croscarmellosesodium, carmellose calcium, crospovidone, alginic acid, sodium alginate,potassium alginate, calcium alginate, an ion exchange resin, aneffervescent system based on food acids and an alkaline carbonatecomponent, clay, talc, starch, pregelatinized starch, sodium starchglycolate, cellulose floc, carboxymethylcellulose,hydroxypropylcellulose, calcium silicate, a metal carbonate, sodiumbicarbonate, calcium citrate, or calcium phosphate.

Still further embodiments of the invention include the active agentadministered in combination with other active such as, for example,adjuvants, protease inhibitors, or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein.

Other embodiments of the present invention include a pharmaceuticalcomposition comprising an effective amount of the active agent and oneor more pharmaceutically acceptable excipient. Other embodiments includea pharmaceutical composition comprising an effective amount ofpharmaceutically-acceptable salts of the active agent. Other embodimentsinclude a pharmaceutical composition comprising an effective amount ofpharmaceutically-acceptable salts of active agent and apharmaceutically-acceptable excipient.

In yet other embodiments, the active agent may be combined with one ormore secondary therapeutic agents. Secondary therapeutic agents myinclude but are not limited to: an anti-platelet agent, an inhibitor ofangiotensin II, an ACE inhibitor, a Ca⁺⁺ channel blocker, an insulinsensitizer, a HMG-CoA reductase inhibitor, a beta blocker, anon-steroidal anti-inflammatory drug, a steroidal anti-inflammatorydrug, peroxisome proliferator-activated receptors (PPAR) modulators, andcombinations thereof.

Pluripotent anti-inflammatory and metabolic modulators andpharmaceutical compositions thereof as described herein may beadministered to subjects to treat a number of both acute and chronicinflammatory and metabolic conditions. In some embodiments, thepluripotent anti-inflammatory and metabolic modulators andpharmaceutical compositions thereof as described herein may be used totreat acute conditions including general inflammation, autoimmunedisease, auto-inflammatory disease, arterial stenosis, organ transplantrejection and burns, and chronic conditions such as, chronic lung injuryand respiratory distress, diabetes, hypertension, obesity, arthritis,neurodegenerative disorders and various skin disorders.

However, in other embodiments, the pluripotent anti-inflammatory andmetabolic modulators and pharmaceutical compositions thereof asdescribed herein may be used to treat any condition having symptomsincluding chronic or acute inflammation, such as, for example,arthritis, lupus, Lyme's disease, gout, sepsis, hyperthermia, ulcers,enterocolitis, osteoporosis, viral or bacterial infections,cytomegalovirus, periodontal disease, glomerulonephritis, sarcoidosis,lung disease, lung inflammation, fibrosis of the lung, asthma, acquiredrespiratory distress syndrome, tobacco induced lung disease, granulomaformation, fibrosis of the liver, graft vs. host disease, postsurgicalinflammation, coronary and peripheral vessel restenosis followingangioplasty, stent placement or bypass graft, coronary artery bypassgraft (CABG), acute and chronic leukemia, B lymphocyte leukemia,neoplastic diseases, arteriosclerosis, atherosclerosis, myocardialinflammation, psoriasis, immunodeficiency, disseminated intravascularcoagulation, systemic sclerosis, amyotrophic lateral sclerosis, multiplesclerosis, Parkinson's disease, Alzheimer's disease, encephalomyelitis,edema, inflammatory bowel disease, hyper IgE syndrome, cancer metastasisor growth, adoptive immune therapy, reperfusion syndrome, radiationburns, alopecia and the like.

The compound of Formula I and pharmaceutical compositions thereof asdescribed herein may be administered to subjects to treat tissueallograft rejection. In other embodiments, the compound of Formula I andpharmaceutical compositions thereof as described herein may beadministered to subjects to prevent or reduce rejection of atransplanted organ. In some embodiments, the compound of Formula I andpharmaceutical compositions thereof as described herein may be used toprolong the survival of a grafted tissue. In some embodiments, thecompound of Formula I and pharmaceutical compositions thereof asdescribed herein may be used to prolong the survival of a transplantedorgan.

EXAMPLES

The following examples contain detailed methods of preparing compoundsof Formula I. These detailed descriptions serve to exemplify the abovegeneral synthetic schemes which form part of the invention. Thesedetailed descriptions are presented for illustrative purposes only andare not intended as a restriction on the scope of the invention. Allparts are by weight and temperatures are in Degrees Celsius unlessotherwise indicated. All compounds showed NMR spectra consistent withtheir assigned structures.

Example 1: 2-Hydroxy-5-(2-Nitroethenyl)Benzoic Acid (SANA)

To a solution of 5-formylsalicylic (1 g, 6.02 mmol) in ethanol (16.5mL), nitromethane (5.5 mL, 0.10 mmol) and ammonium acetate (1.39 g,18.06 mmol) were added. The reaction mixture is heated at 60° C. for h,allowed to cool to room temperature and put in refrigerator for 15minutes. Formed orange precipitate was filtered off and dissolved inwater (ca. 250 mL). Solution was acidified with concentrated HCl (ca. 10drops) until total precipitation. Formed yellow solid was filtered offand dried in vacuo. Yield: 1.18 g (93%).

1H NMR (acetone-d₆): δ=8.35 (d, J=2.3 Hz, 1H), 8.14 (d, J=13.7, 1H),8.06 (dd, J=8.7 2.3 Hz, 1H), 7.99 (d, J=13.7 Hz, 1H), 7.11 (d, J=8.7 Hz,1H). 13C NMR (acetone-d₆): δ=171.09, 164.66, 137.94, 136.51, 135.94,133.21, 121.95, 118.51, 113.08

Biologic Activity

The following methods described are used in order to demonstratebiological activity and therapeutic use, and should not to be construedin any way as limiting the scope of the invention.

In Vitro Activity

As shown in FIG. 1, SANA (100 mM) in phosphate buffer 100 mM, pH 7.4 wasincubated with β-mercaptoethanol (BME) (1 mM) or Glutathione (GSH) (1mM) and the reaction was followed spectrophotometrically (1 scan every 1min for up to 15 min). UV-visible spectra was acquired by a Varian Cary50 Bio. Scans were taken every min up to 15 min.

The reaction between SANA and BME or GSH showed increased absorption atthe 360 nm wavelength. The increase at 360 nm demonstrates adductformation between SANA and BME or GSH.

In FIG. 2, it is shown that the reaction between SANA and BME wasdetermined to be a second order rate constant. Stopped-flow kineticmeasurements were performed using an Rx 2000 stopped flow analyzer(Applied Photophysics). Mixtures of 150 μL NAT×0 (25 μM) and solutionsof BME at 0.54 mM, 1.09 mM, 1.64 mM, 2.18 mM, and 2.73 mMconcentrations.

The reaction was monitored by following the absorbance at 260 nm andplots were fitted to a simple exponential decay function using Originlabsoftware (version 8.0.). The observed pseudo first order constant ateach concentration of BME was extracted from the equation and plottedagainst the concentration of BME. The second rate constant of thereaction is derived from the slope of the curve and was 182±6 M⁻¹s⁻¹.All experiments were carried out at 25° C. by triplicate.

Nuclear factor kappa B (NF-κB) represents a family of pro-inflammatorytranscription factors, present in all eukaryotic cells, which regulateinducible expression of wide ranging genes involved in immune responsesand cell-cycle regulation. Activation of NF-κB is accompanied by nucleartranslocation of NF-κB. Accordingly, FIG. 3 illustrates the lack ofnuclear translocation of NF-κB in the presence of SANA to furtherdemonstrate its anti-inflammatory effects. Specifically, FIG. 3illustrates immunofluorescence and epifluorescence microscopy analysisshowing the effect of SANA on LPS-induced NF-κB/p65 subcellularlocalization in THP-1 macrophages. Unexpectedly, cells treated with SANAat a concentration of 0.1 mM, 2 hs before activation with LPS (1 ug/mL)showed the same effect as cells treated with salicylic acid at 1 mM.This data indicates that SANA was significantly more potent thansalicylic acid.

FIG. 4 shows the inhibition of NF-kB-dependent gene expression in humanmacrophages by SANA. THP-1 cells were differentiated into macrophages.Cells were then treated with SANA (100 and 200 uM) or Salicylic acid(SA: 100 and 200 uM) for 2 hours. Cells were then stimulated with LPS (1mg/mL, 3 hours). mRNA was extracted and IL-6, TNF-a and MCP-1 foldchange gene expression over control were quantified by qPCR.Interestingly, FIG. 4 shows that when uses at the same concentration, SAwas not able to inhibit NF-kB dependent gene expression in these cells.

In FIG. 5 murine RAW 264.7 macrophages were treated with/without SANA(0, 50; 100 and 200 uM, 2 hours) or SA (1 mM) to see the potentialinhibition of NF-kB dependent gene expression in murine macrophages.Cells were then stimulated with LPS (50 ng/mL, 16 hours). Supernatantswere collected and IL-6 was measured by ELISA. We applied one-way ANOVAstatistic test with Bonferroni post-host. (*):p<0.05 compared toLPS-DMSO. FIG. 5. shows that SANA is a more potent inhibitor of NF-kBdependent gene expression in these cells than SA.

FIGS. 6 and 7 show induction of phase two enzymes Nrf2/Keap 1-dependentgene expression by SANA but not by SA. Hep G2 cells were treated withSANA (0.1 mM) or Salicylic Acid (0.2 and 5 mM) for five hours. mRNA wasextracted from the cells and was measured HO-1, GCLM and NQO1 geneexpression by qPCR.

In FIGS. 8 and 9 show the inhibition of inflammasome in THP-1 cellsdifferentiated into macrophages (PMA 200 nM, 48 hs.) by SANA but not bySA when applied together with the first (FIG. 8) or the second (FIG. 9)signal. In FIG. 8 cells were treated with Salicylic acid (0.25 mM) orSANA (0.05; 0.125 and 0.25) together with LPS stimulation. The cellswere stimulated with LPS (250 ng/mL, 3 hs.) and then with ATP (5 mM, 45minutes). Supernatant was collected and IL-1b measured by ELISA. Cellviability was assessed by MTT assay. The values are showed as mean±SD.

In FIG. 9 THP-1 cells were differentiated into macrophages with PMA (200nM, 48 hs.). Cells were stimulated with LPS (250 ng/mL, 3 hs.) and thenwith ATP (5 mM, 45 minutes). Together with ATP treatment, cells werethen treated with NAT×ME (10 uM), Salicylic acid (0.25 mM) or SANA(0.05; 0.125 and 0.25 mM). Supernatant was collected and IL-1b secretionwas measured by ELISA. Cell viability was assessed by the MTT assay. Thevalues are showed as mean±SD. FIGS. 8 and 9 show that SAN is a potentinhibitor of the inflammasome when applied with the first or secondsignal, whereas SA cannot inhibit this potent pro-inflammatory cellularpathway.

In Vivo Activity

FIG. 10 shows the effect of SANA on AMPK phosphorylation in vivo.C57BL/6 mice were treated with SANA (200 mg/kg, gavage) or PBS (Na₂HPO₄76 mM; NaH₂PO₄ 24 mM; NaCl 17 mM pH 7.4. One hour after administration,livers were extracted and then were homogenized into NETN lysis buffer.pAMPK was then detected by Western blot. Each condition was studied intwo mice (n=2).

In FIG. 11 C57BL/6 mice were treated with SANA or SA (100, 200 and 300mg/kg, intraperitoneal) or PBS, pH 7.4 (Na₂HPO₄ 76 mM; NaH₂PO₄ 24 mM;NaCl 17 mM). Two hours after administration, livers were extracted andthen were homogenized into NETN lysis buffer. pAMPK was then detected bywestern blot. Each condition was studied in one mouse (n=1).

In FIG. 12 C57BL/6 mice were treated with SANA up to 400 mg/kgintraperitoneally or PBS, pH 7.4 (Na₂HPO₄ 76 mM; NaH₂PO₄ 24 mM; NaCl 17mM). Two hours after administration, livers were extracted and then werehomogenized into NETN lysis buffer. pAMPK was then detected by westernblot. Each condition was studied in one mouse (n=1).

FIG. 13 shows that SANA decreases LPS-induced Il-1b secretion into theperitoneum in vivo.

FIG. 14 shows that SANA reverses insulin resistance in HFD-induced obesemice. Mice under HFD for up to 7 months (mean weight around 40 gr.) weretreated with SANA (100 mg/kg; gavage) or phosphate buffer (control)every day during four weeks. The glucose tolerance test were run as perwell-known standard procedures.

FIG. 15 shows that SANA unexpectedly does not inhibit GAPDH activitywhile that is commonly observed with nitroalkenes.

As shown in FIG. 16, administering SANA to subjects demonstratedprolonged survival of grafted skin as compared to a control group.C57BL/6 female mice were grafted with skin from the tails of C57BL/6male mice. The skin graft is a small square (1 cm²) and it was implantedonto the left subscapular region of the female mice. From one day beforethe transplantation until 15 days after it, the mice were treated dailywith SANA (100 mg/kg, by oral gavage; SANA group) or vehicle (Controlgroup). The vehicle used was a solution of Carboxy-Methyl-cellulose 0.5%m/v and Tween 80 0.5% v/v. At the end of the 15th day of administration,allografts were checked every 48/72 hours to assess the condition of thegrafted skin. Rejection was diagnosed clinically when the allograft lostmore than 50% of its size and/or more than 10% of allograft wasnecrotic.

Results from FIG. 17 indicate that treatment with SANA providedsignificant reduction in skin allograft rejection compared to salicylicacid. The study followed the protocol described above for FIG. 1, butfurther included a Salicylate group. C57BL/6 female mice were graftedwith skin from the tails of C57BL/6 male mice. The skin graft is a smallsquare (1 cm²) and it was implanted onto the left subscapular region ofthe female mice. From one day before the transplantation until 15 daysafter it, the mice were treated daily with Salicylate (100 mg/kg, byoral gavage; Salicylate group), SANA (100 mg/kg, by oral gavage; SANAgroup) or vehicle (Control group). The vehicle used was a solution ofCarboxy-Methyl-cellulose 0.5% m/v and Tween 80 0.5% v/v. At the end ofthe 15th day of administration, allografts were checked every 48/72hours to assess the condition of the grafted skin. Rejection wasdiagnosed clinically when the allograft lost more than 50% of its sizeand/or more than 10%¹ of allograft was necrotic.

NON-PATENT CITATIONS

-   1. Manabe, I. “Chronic Inflammation Links Cardiovascular, Metabolic    and Renal Diseases,” Circ J. 75(12):2739-48 (2011).-   2. Antman, E M et al. “Use of nonsteroidal antiinflammatory drugs:    an update for clinicians: a scientific statement from the American    Heart Association,”Circulation 115(12):1634-42 (Mar. 27, 2007).

What is claimed is:
 1. A method of treating acute and chronicinflammatory conditions comprising administering to a subject in needthereof a therapeutically effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,further comprising administering a secondary therapeutic agent.
 3. Themethod of claim 2, wherein the secondary therapeutic agent is selectedfrom the group consisting of an anti-platelet agent, an inhibitor ofangiotensin II, an ACE inhibitor, a Ca⁺⁺ channel blocker, an insulinsensitizer, a HMG-CoA reductase inhibitor, a beta blocker, anon-steroidal anti-inflammatory drug, a steroidal anti-inflammatorydrug, peroxisome proliferator-activated receptors (PPAR) modulators, andcombinations thereof.
 4. A method of treating organ transplant rejectioncomprising administering to a subject in need thereof an effectiveamount of a compound of Formula I:


5. The method of claim 4, wherein the organ transplant is a skinallograft.
 6. A method of treating organ transplant rejection in asubject comprising administering to said subject a pharmaceuticalcomposition comprised of an effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, and a carrier.
 7. Themethod of claim 6, wherein the organ transplant is a skin allograft.